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Am J Physiol Gastrointest Liver Physiol 290: G1194-G1202, 2006. First published September 29, 2005; doi:10.1152/ajpgi.00345.2005
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HORMONES AND SIGNALING

Occupation of {alpha}vbeta3-integrin by endogenous ligands modulates IGF-I receptor activation and proliferation of human intestinal smooth muscle

John F. Kuemmerle

Departments of Medicine and Physiology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia

Submitted 26 July 2005 ; accepted in final form 26 September 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We have previously shown that endogenous IGF-I regulates growth of human intestinal smooth muscle cells by stimulating proliferation and inhibiting apoptosis. In active Crohn's disease, expression of IGF-I and the {alpha}vbeta3-integrin receptor ligands fibronectin and vitronectin is increased. The aim of the present study was to determine whether occupation of the {alpha}vbeta3-receptor influences IGF-I receptor tyrosine kinase activation and function in human intestinal smooth muscle cells. In untreated cells, IGF-I elicited time-dependent tyrosine phosphorylation of its cognate receptor that was maximal within 2 min and sustained for 30 min. In the presence of the {alpha}vbeta3-ligand fibronectin, IGF-I-stimulated IGF-I receptor activation was augmented. Conversely, in the presence of the {alpha}vbeta3-specific disintegrin echistatin, IGF-I-stimulated IGF-I receptor tyrosine kinase phosphorylation was inhibited. IGF-I-stimulated IGF-I receptor activation was accompanied by recruitment of the adapter protein IRS-1, activation of Erk1/2, p70S6 kinase, and proliferation. These effects were augmented by fibronectin and attenuated by echistatin. IGF-I also elicited time-dependent recruitment of protein tyrosine phosphatase SHP-2 that coincided with dephosphorylation of the tyrosine phosphorylated IGF-I receptor tyrosine kinase. The {alpha}vbeta3-disintegrin echistatin accelerated the rate of SHP-2 recruitment and deactivation of the IGF-I receptor tyrosine kinase. The results show that occupancy of the {alpha}vbeta3-integrin receptor modulates IGF-I-induced IGF-I receptor activation and function in human intestinal muscle cells. We hypothesize that the concomitant increases in the expression of {alpha}vbeta3-ligands and of IGF-I in active Crohn's disease may contribute to muscle hyperplasia and stricture formation by acting in concert to augment IGF-I-stimulated IGF-I receptor tyrosine kinase activity and IGF-I-mediated muscle cell growth.

echistatin; vitronectin; fibronectin; SHP-2; Crohn’s disease

INTEGRINS ARE A FAMILY OF transmembrane proteins comprised of 18 {alpha}-subunits and 8 beta-subunits that heterodimerize to form the 24 known integrin pairs (9). A wide variety of integrin ligands have been described that bind to specific integrins and together participate in outside-in and inside-out integrin signaling (17). Although integrin expression, in general, is widespread throughout the body, the expression of specific integrins and their preferred ligands is tissue specific. This confers one level of specificity to an otherwise ubiquitously expressed family of proteins and is fundamental to their ability to regulate cellular events. Smooth muscle of visceral and vascular types express the {alpha}1beta1-, {alpha}5beta1-, {alpha}vbeta3-, {alpha}2beta1-, {alpha}3beta1-, {alpha}5beta1-, {alpha}Vbeta1-, {alpha}Vbeta5-, and {alpha}1beta6-integrins (28). Unlike receptors with intrinsic tyrosine kinase-containing activity in response to their ligands, integrin receptors undergo a conformational change that alters their activation state (5). Phosphorylation of cytoplasmic domains of integrin beta-subunits results in the association of signaling proteins (25, 36).

Experimental evidence in cells that were detached and reattached to extracellular matrix proteins has shown that integrins can modulate growth factor-stimulated receptor activation and thereby regulate growth factor-stimulated cell adhesion and cell motility (38). Clemmons et al. (7) and Zheng and Clemmons (37) have shown that in porcine vascular smooth muscle, {alpha}vbeta3-integrin (the cognate vitronectin receptor) modulates IGF-I-stimulated smooth muscle motility and proliferation in stably attached cells in vitro without being subjected to detachment and reattachment. The {alpha}vbeta3-integrin has also been shown to play a key role in the regulation of IGF-I-mediated vascular smooth muscle growth during atheroma formation in vivo (29). The muscle hyperplasia that accompanies atheroma development and contributes to arterial narrowing can be prevented by a neutralizing antibody to {alpha}vbeta3-integrin (29). Integrin and IGF-I-modulated uterine smooth muscle growth has also been implicated as a factor in leiomyoma formation (4, 33).

In cultured porcine vascular smooth muscle cells, three transmembrane proteins, the {alpha}vbeta3-integrin, tyrosine phosphatase SHP-2 substrate 1 (SHPS-1), and the IGF-I receptor (IGF-IR) tyrosine kinase, jointly regulate cellular responses to IGF-I (8). Acting in concert, these three proteins regulate the recruitment of the SH2 domain-containing protein tyrosine phosphatase SHP-2 to the IGF-IR tyrosine kinase (27). Interplay between the {alpha}vbeta3-integrin and growth factor receptors is a common theme, and its modulation of PDGF, VEGF, and insulin signaling has been demonstrated (32). The EGF receptor has been shown to interact directly with {alpha}5beta1, {alpha}6beta4, and {alpha}Vbeta1 integrins (13). The activation of PDGF, FGF, insulin, and EGF receptor tyrosine kinases, in addition to the IGF-IR tyrosine kinase, is modulated by the protein tyrosine phosphatase SHP-2.

Patients with active Crohn's Disease have elevated plasma levels of fibronectin, a ligand of {alpha}vbeta3-integrin (2). In regions of inflammation and stricturing, the numbers of mast cells resident in the muscularis propria are higher than in the muscle layer of uninflamed intestine. Although immunoreactive laminin colocalized with markers of tissue mast cells, immunoreactive fibronectin or vitronectin did not (14). Neither the expression of {alpha}vbeta3-integrin and of its ligands, fibronectin and vitronectin, by intestinal smooth muscle nor the role of this system in regulating muscle cells proliferation in response to IGF-I has been examined.

This paper shows that human intestinal smooth muscle cells express the {alpha}V- and beta3 integrin subunits and the {alpha}vbeta3-integrin ligands vitronectin and fibronectin in vivo. Expression of all four remains constant when intestinal smooth muscle cells are cultured. IGF-I stimulates time-dependent phosphorylation of the IGF-IR tyrosine kinase, recruitment, and phosphorylation of insulin receptor substrate-1 (IRS-1) and the downstream kinases Erk1/2 and p70S6 kinase phosphorylation, which jointly stimulate muscle cell proliferation (20). SH2-domain containing protein tyrosine phosphatase SHP-2 is recruited in a time-dependent fashion to the activated IGF-IR tyrosine kinase. SHP-2 recruitment coincides in time with dephosphorylation of the IGF-IR tyrosine kinase. The {alpha}vbeta3-integrin ligand fibronectin augments IGF-I-stimulated IGF-IR tyrosine kinase phosphorylation, Erk1/2 and p70S6 kinase phosphorylation, and proliferation. In contrast, the {alpha}vbeta3-selective antagonist echistatin diminished IGF-I-stimulated IGF-IR phosphorylation, recruitment, and phosphorylation of IRS-1, phosphorylation of Erk1/2 and p70S6 kinase, and muscle cell proliferation. The rate of IGF-I-stimulated SHP-2 recruitment to the IGF-IR in the presence of echistatin was accelerated. The results implied that the activation state of {alpha}vbeta3-integrin is regulated by endogenous integrin ligands and modulates IGF-I-stimulated receptor activation, signaling pathways, and proliferation in human intestinal smooth muscle cells.

The potential clinical significance of this mechanism has relevance in the setting of chronic intestinal inflammation when levels of the {alpha}vbeta3-integrin ligands vitronectin and fibronectin are increased and could act in concert with the observed increased IGF-I levels that augment intestinal muscle cell proliferation and inhibition of apoptosis leading to hyperplasia of the muscularis propria and stricture formation.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
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Culture of smooth muscle cells isolated from normal human jejunum.

Muscle cells were isolated and cultured from the circular muscle layer of human jejunum as described previously (19, 20). Briefly, 4- to 5-cm segments of normal intestine were obtained from patients undergoing surgery according to a protocol approved by the Virginia Commonwealth University Institutional Review Board. After the segments along the mesenteric border were opened, the mucosa was dissected away and the remaining muscle layer was cut into 2 x 2-cm strips. Slices were obtained separately from the circular layer using a Stadie-Riggs tissue slicer. The slices were incubated overnight at 37°C in 20 ml of DMEM plus 10% fetal bovine serum (DMEM-10) containing penicillin 200 U/ml, streptomycin 200 µg/ml, gentamycin 100 µg/ml, and 2 µg/ml amphotericin B to which was added 0.0375% collagenase (type II) and 0.1% soybean trypsin inhibitor. Muscle cells dispersed from the circular layer were harvested by filtration through 500-µm Nitex mesh and centrifugation at 150 g for 5 min. Cells were resuspended and washed twice by centrifugation at 150 g for 5 min. After resuspension in DMEM-10 containing the same antibiotics, the cells were plated at a concentration of 5 x 105 cells per milliliter as determined by counting in a hemocytometer. Cultures were incubated in a 10% CO2 environment at 37°C. DMEM-10 medium was replaced every 3 days until the cells reached confluence.

Primary cultures of muscle cells were passaged on reaching confluence and used in the first passage. We have previously shown that these cells express a phenotype characteristic of intestinal smooth muscle as determined by immunostaining for smooth muscle markers (clone HM 19/2, 5 µg/ml) and the expression of {gamma}-enteric actin (34). Epithelial cells, endothelial cells, neurons, and interstitial cells of Cajal are not detected in these cultures.

Immunoprecipitation of IGF-IR.

IGF-IRs were immunoprecipitated as previously described (21). Briefly, confluent smooth muscle cells were rendered quiescent by incubation in serum-free DMEM for 24 h. Cells were incubated with the {alpha}vbeta3-integrin ligand fibronectin (5 µg/cm2 cultureware surface area), the {alpha}vbeta3-selective disintegrin, echistatin (100 nM), or vehicle for the final 12 h (7). Cells were then stimulated with 100 nM IGF-I for periods of time from 0 to 30 min. The reaction was terminated by washing twice with ice-cold PBS. Cell lysates were prepared in an immunoprecipitation buffer consisting of (in mM) 50 Tris·HCl (pH 7.5), 150 NaCl, 50 NaF, 1 Na orthovanadate, 1 dithiothreitol, 1 phenylmethylsulfonyl fluoride, and 0.5% Nonidet P-40 to which was added 1 µg/ml leupeptin, 1 µg/ml pepstatin A, and 1 µg/ml aprotinin. The resulting lysates were clarified by centrifugation at 14,000 g for 10 min at 4°C. The lysates were precleared by incubation with protein A agarose beads for 1 h at 4°C. Samples containing equal amounts of protein (1 mg) were incubated for 2 h at 4°C with 2 µg rabbit anti-IGF-IR beta-subunit. The incubation continued overnight at 4°C after the addition of 10 µl protein A agarose beads. The immune complex-agarose beads were washed three times with ice-cold immunoprecipitation buffer and twice with ice-cold kinase assay buffer. After being washed, the immune complex-agarose beads were resuspended in 25 µl sample buffer, boiled for 5 min, and then loaded onto a 7.5% polyacrylamide gel, and the proteins were separated by SDS-PAGE. Tyrosine-phosphorylated IGF-IR and coimmunoprecipated IRS-1 or the protein tyrosine phosphatase SHP-2 were identified by immunoblot analysis.

Western blot analysis.

Analysis of proteins and phosphorylated proteins was performed by Western blot analysis using standard methods (18, 20–22). Briefly, confluent muscle cells were rendered quiescent by incubation for 24 h in serum-free medium. The cells were stimulated with recombinant human IGF-I for periods of time from 0 to 30 min. The reaction was terminated by two rapid washes in ice-cold PBS. Sample buffer was added to cells to lyse the cells or added to immunoprecipitated proteins. After being boiled for 5 min, samples adjusted to contain equal amounts of total protein before immunoprecipitation or samples adjusted to contain equal amounts of total protein after being directly lysed were separated with SDS-PAGE under denaturing conditions. After the proteins were electrotransferred to nitrocellulose, the membranes were incubated overnight with specific antibodies recognizing the proteins of interest (dilution): {alpha}V (1:500), beta3 (1:500), fibronectin (1:1,000), vitronectin (1:1,000), phosphotyrosine (1:1,000), SHP-2 (1:1,000), IRS-1 (1:1,000), phospho-IRS-1(Tyr632) (1:1,000), Erk1/2 (1:1,000), phospho-Erk1/2(Thr202/Tyr204) (1:2,000), p70S6 kinase (1:1,000), or phospho-p70S6 kinase(Ser389) (1:1,000). When appropriate, nitrocellulose membranes were stripped and reblotted to determine levels of total (phosphorylated + nonphosphorylated) protein. Bands of interest were visualized with enhanced chemiluminescence using a FluoChem 8800 (Alpha Innotech, San Leandro, CA), and the resulting digital images were quantified using AlphaEaseFC version 3.1.2 software.

[3H]thymidine incorporation assay.

Proliferation of smooth muscle cells in culture was measured by the incorporation of [3H]thymidine as described previously (19, 22). Briefly, the cells were washed free of serum and incubated for 24 h in serum-free DMEM in the presence or absence of various test agents. During the final 4 h of this incubation period, 1 µCi/ml [3H]thymidine was added to the medium. [3H]thymidine incorporation into the perchloric acid extractable pool was used as a measure of DNA synthesis.

Measurement of protein content.

The protein content of cell lysates was measured using the Bio-Rad DC protein assay kit according to manufacturer's direction. Samples were adjusted to provide aliquots of equal protein content before in vitro kinase assay or Western blot analysis.

Statistical analysis.

Values given represent the means ± SE of number of experiments (n) on cells derived from separate primary cultures. Statistical significance was tested by Student's t-test for either paired or unpaired data as was appropriate. Densitometric values for protein bands of phosphorylated signaling intermediates were reported in arbitrary units above background values after normalization to total protein levels. Bands of interest were visualized with enhanced chemiluminescence using a FluoChem 8800 (Alpha Innotech, San Leandro, CA), and the resulting digital images were quantified using AlphaEaseFC version 3.1.2 software.

Materials.

Recombinant human IGF-I was obtained from Austral Biologicals (San Ramon, CA). Collagenase and soybean trypsin inhibitor were obtained from Worthington Biochemical (Freehold, NJ). HEPES was obtained from Research Organics (Cleveland, OH). DMEM and Hanks' balanced salt solution were obtained from Mediatech (Herndon, VA). Fetal bovine serum was obtained from Summit Biotechnologies (Fort Collins, CO). Rabbit polyclonal antibodies to p-IRS-1(Tyr632), IRS-1, and p70S6 kinase, mouse monoclonal antibodies to phosphotyrosine (p-Tyr100), p-Erk1/2(Thr202/Tyr204), and Erk1/2, anti-rabbit IgG-HRP, and anti-mouse-HRP were obtained from Cell Signaling Technology (Beverly, MA). Rabbit polyclonal antibody to p-p70S6 kinase(Ser389) was obtained from Upstate Biotechnology (Lake Placid, NY). Rabbit polyclonal antibody to beta3 was obtained from Biosource International (Camarillo, CA). Human fibronectin and rabbit polyclonal antibody to {alpha}V were obtained from Becton-Dickinson (Franklin Lakes, NJ). Western blotting materials and DC protein assay kit were obtained from BioRad Laboratories (Hercules, CA). Mouse-rat monoclonal antibody to smooth muscle (HM19/2) from Biogenesis (Sandown, NH). Plastic cultureware was obtained from Corning (Corning, NY). Echistatin and all other chemicals were obtained from Sigma (St. Louis, MO).


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Expression of {alpha}V- and beta3-integrin subunits in fresh and cultured muscle cells.

The expression of the {alpha}V- and beta3-integrin subunits was examined in human intestinal circular smooth muscle in vivo and their continued expression confirmed in cultured intestinal smooth muscle cells in vitro. Cell lysates were prepared from fresh and cultured smooth muscle cells and adjusted to contain equal concentrations of total protein. Western blot analysis of these cell lysates using an antibody selective for the {alpha}V-integrin subunit identified a protein of 125-kDa size, corresponding to the known size of the {alpha}V-integrin subunit (Fig. 1A). Western blot analysis of cell lysates from fresh and cultured cells using a selective beta3-integrin subunit antibody identified a protein of 105-kDa size, corresponding to the know size of the beta3-integrin subunit (Fig. 1B). Similar levels of integrin subunit expression were present in fresh muscle cells compared with cultured muscle cells based on total cell protein.


Figure 1
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  		Fig. 1. Expression of {alpha}V- and beta3-integrin subunits in fresh and cultured human intestinal smooth muscle. {alpha}V (A) and beta3-integrin (B) subunits were identified in freshly dispersed human intestinal smooth muscle cells. Their levels were similar in cultured intestinal smooth muscle cells. Cell lysates were prepared from freshly dispersed intestinal muscle cells and from cultured muscle cells and lysates containing equal amounts of total protein were subjected to immunoblot analysis using antibodies specifically recognizing the {alpha}V- and beta3-integrin subunits.

 
Expression of {alpha}vbeta3-integrin ligands in fresh and cultured muscle cells.

Two important ligands of the {alpha}vbeta3-integrin, the cognate vitronectin receptor, are vitronectin and fibronectin. The expression of vitronectin and of fibronectin in human intestinal smooth muscle cells in vivo and in cultured smooth muscle in vitro was therefore examined. Western blot analysis of cell lysates prepared from fresh tissue and from cultured cells using a fibronectin-specific antibody identified a protein of 90-kDa size, similar to the predicted size of fibronectin and coinciding with authentic fibronectin (Fig. 2A). Western blot analysis of cell lysates prepared from fresh tissue and from cultured cells using a vitronectin specific antibody identified a protein of 60-kDa size, similar to the predicted size of vitronectin and coinciding with authentic vitronectin (Fig. 2B). Similar levels of fibronectin and vitronectin were present in fresh muscle cells compared with cultured muscle cells.


Figure 2
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  		Fig. 2. Expression of vitronectin and fibronectin in fresh and cultured human intestinal smooth muscle. The {alpha}vbeta3-integrin ligands fibronectin (A) and vitronectin (B) were identified in freshly dispersed human intestinal smooth muscle cells. Their levels were similar in cultured intestinal smooth muscle cells. Cell lysates were prepared from freshly dispersed intestinal muscle cells and from cultured muscle cells, and lysates containing equal amounts of total protein were subjected to immunoblot analysis using antibodies specifically recognizing vitronectin and fibronectin.

 
Role of {alpha}vbeta3-integrin in IGF-IR activation.

The effect of {alpha}vbeta3-integrin occupancy by its endogenous ligands on IGF-I-stimulated IGF-IR activation was examined in cultured human intestinal smooth muscle cells. Muscle cells were rendered quiescent by incubation in serum-free DMEM for 24 h. During the final 12 h of the incubation, the cells were exposed to either the {alpha}vbeta3-integrin ligand fibronectin (5 µg/cm2 cultureware surface area) or the {alpha}vbeta3-integrin specific antagonist echistatin (100 nM). Echistatin, a selective {alpha}vbeta3-antagonist, binds to the cytoplasmic portion of the beta3-subunit of {alpha}vbeta3-integrin and disrupts its function. At the end of the 24 h, cells were stimulated with a maximally effective concentration of IGF-I (100 nM) for periods of 0–30 min. Immunoprecipitation and immunoblot analysis of samples containing equal amounts of protein showed that IGF-I elicited phosphorylation of the IGF-IR that was rapid, attained a maximum of 341 ± 46% above basal within 2 min, and within 30 min declined to 190 ± 35% above basal, i.e., IGF-IR dephosphorylation had occurred (Fig. 3). In the presence of fibronectin, IGF-I-induced IGF-IR phosphorylation was increased at all time periods. The results implied that the extent of {alpha}vbeta3-integrin occupancy influences the extent of IGF-IR activation.


Figure 3
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  		Fig. 3. IGF-I-stimulated IGF-I receptor (IGF-IR) tyrosine kinase phosphorylation is modulated by the {alpha}vbeta3-integrin. A: representative immunoblots of time-dependent IGF-I-stimulated IGF-IR tyrosine kinase phosphorylation in control, fibronectin-treated, and echistatin-treated human intestinal smooth muscle cells. IP, immunoprecipitate; IB, immunoblot. B: IGF-I elicits time-dependent IGF-IR tyrosine kinase phosphorylation that is maximal within 2 min. In the presence of the {alpha}vbeta3-integrin ligand fibronectin, the IGF-I-stimulated IGF-IR tyrosine kinase phosphorylation is enhanced at all time periods. In the presence of the {alpha}vbeta3-integrin antagonist echistatin, IGF-I-stimulated IGF-IR tyrosine kinase phosphorylation is inhibited at all time periods. Quiescent muscle cells were treated for 0–30 min with 100 nM IGF-I. Fibronectin (5 µg/cm2) was used as a ligand of the {alpha}vbeta3-integrin, and echistatin (100 µM) was used as a selective {alpha}vbeta3-integrin antagonist. IGF-IR tyrosine kinase phosphorylation was determined in IGF-IR IPs by phosphotyrosine IB. Results were expressed in relative densitometric values normalized to total (phosphorylated plus unphosphorylated) IGF-IR levels. Values represent the means ± SE of 3–5 separate experiments. *P < 0.05 vs. control.

 
In the presence of echistatin, IGF-I-stimulated IGF-IR phosphorylation at 2 min was inhibited by 79 ± 6% and was abolished at longer time periods (Fig. 3). The ability of the {alpha}vbeta3-receptor antagonist echistatin to alter the kinetics of IGF-I-stimulated IGF-IR receptor phosphorylation implied that endogenous {alpha}vbeta3-integrin activity contributes to the regulation of extent and duration of IGF-IR phosphorylation. The SH2 domain-containing tyrosine phosphatase SHP-2 is recruited in other cell types to the activated IGF-IR tyrosine kinase and regulates is activity by mediating receptor tyrosine kinase dephosphorylation (1, 27).

Recruitment of tyrosine phosphatase SHP-2 to the activated IGF-IR.

The time course of SHP-2 recruitment to the activated IGF-IR was examined in quiescent muscle cells and in cells treated with the {alpha}vbeta3-integrin antagonist echistatin. Incubation of quiescent muscle cells with 100 nM IGF-I for periods of 0–30 min elicited time-dependent association of SHP-2 with the IGF-I-activated IGF-IR tyrosine kinase (Fig. 4). The association was gradual and attained a maximum of 200 ± 30% above basal after 30 min. In cells treated with the {alpha}vbeta3-integrin antagonist, the time course of SHP-2 association was accelerated. In the presence of echistatin, maximal SHP-2 association with the IGF-IR of 255 ± 27% above basal occurred within 5 min and had declined to basal levels after 30 min. The recruitment of SHP-2 to the activated IGF-IR, in both naïve and echistatin-treated cells, coincided in time with IGF-IR dephosphorylation, suggesting that the SHP-2 phosphatase may be responsible for inactivation of the IGF-IR that follows IGF-I stimulation. The ability of the {alpha}vbeta3-antagonist echistatin to alter the kinetics of IGF-I-stimulated IGF-IR phosphorylation supported the hypothesis that endogenous {alpha}vbeta3-integrin ligands regulated the extent and duration of IGF-IR activity via SHP-2 recruitment and receptor tyrosine kinase dephosphorylation.


Figure 4
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  		Fig. 4. IGF-I-stimulated SHP-2 recruitment to the IGF-IR is modulated by {alpha}vbeta3-integrin. A: representative immunoblots of the time-dependent recruitment of SHP-2 to the IGF-IR in control and echistatin-treated human intestinal muscle cells. B: IGF-I stimulates time-dependent recruitment of SHP-2 phosphatase to the IGF-IR tyrosine kinase. In the presence of the {alpha}vbeta3-integrin antagonist echistatin, SHP-2 phosphatase recruitment occurs early and declines rapidly. Quiescent muscle cells were treated for 0–30 min with 100 nM IGF-I in the presence and absence of 100 nM echistatin. SHP-2 phosphatase recruitment was measured using a coimmunoprecipitation approach in IGF-IR tyrosine kinase immunopreciptates by immunoblotting for SHP-2 phosphatase. Results were expressed in relative densitometric units and normalized to total IGF-IR levels. Values represent the means ± SE of 4 separate experiments. *P < 0.05 vs. control.

 
Role of {alpha}vbeta3 in recruitment of IRS-1 to the IGF-IR.

The involvement of {alpha}vbeta3-integrin on downstream signaling emanating from the activated IGF-IR tyrosine kinase was also investigated. IRS-1 is a principle substrate of the activated IGF-IR. Accordingly, the ability of IGF-I to stimulate the recruitment of IRS-1 to the activated IGF-IR and the role of {alpha}vbeta3-integrin in regulating this process was examined using a coimmunoprecipitation approach. Quiescent muscle cells or muscle cells incubated with echistatin were treated with 100 nM IGF-I for periods of time from 0 to 30 min. IGF-I elicited a prompt association of IRS-1 with the IGF-IR that was maximal within 2 min, 272 ± 16% above basal, and that was sustained at lower levels for up to 30 min (Fig. 5). The 2-min peak in association of IRS-1 and the IGF-IR corresponded to the time of maximal IGF-IR phosphorylation stimulated by IGF-I. In the presence of echistatin, the recruitment of IRS-1 to the activated IGF-IR at the 2-min maximum was inhibited by 87 ± 8% (Fig. 5).


Figure 5
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  		Fig. 5. IGF-I-stimulated IRS-1 recruitment to the IGF-IR is modulated by {alpha}vbeta3-integrin. A: representative immunoblots of the time-dependent recruitment of IRS-1 to the IGF-IR in control and echistatin-treated human intestinal muscle cells. B: IGF-I stimulates time-dependent recruitment of IRS-1 to the IGF-IR tyrosine kinase that is inhibited in the presence of the {alpha}vbeta3-integrin antagonist echistatin. Quiescent muscle cells were treated for 0–30 min with 100 nM IGF-I in the presence and absence of 100 nM echistatin. IRS-1 recruitment was measured using a coimmunoprecipitation approach in IGF-IR tyrosine kinase immunopreciptates by immunoblotting for IRS-1. Results were expressed in relative densitometric units and normalized to total IGF-IR levels. Values represent the means ± SE of 4 separate experiments. *P < 0.05 vs. control.

 
Role of {alpha}vbeta3 in IGF-I stimulated IRS-1 phosphorylation.

The IRS-1 protein possesses ~30 potential tyrosine, serine, and threonine phosphorylation sites. After the recruitment of IRS-1 to the activated IGF-IR, IRS-1 contains numerous potential tyrosine phosphorylation sites, including Tyr-612 and Tyr-632, that result in its activation and its ability to activate its downstream kinases, p85-PI 3-kinase and Grb2, which lead to p70S6 kinase and Erk1/2 activation, respectively (15). Subsequent phosphorylation of IRS-1 on serine residues cause its inactivation (11, 12, 31). Because IRS-1(Tyr632) is specifically phosphorylated by the activated IGF-IR, this was used as a measure of IGF-IR-mediated IRS-1 activation.

Incubation of quiescent muscle cells for periods of time from 0 to 30 min with 100 nM IGF-I elicited time-dependent IRS-1(Tyr632) phosphorylation (Fig. 6). IGF-I-induced IRS-1 phosphorylation was prompt, attaining a maximum of 290 ± 26% above basal within 2 min, was sustained for 10 min before declining after 30 min. In cells treated with the {alpha}vbeta3-integrin antagonist echistatin, the ability of IGF-I to elicit IRS-1(Tyr632) phosphorylation at 2 min was inhibited by 85 ± 9% and abolished at longer time points (Fig. 6).


Figure 6
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  		Fig. 6. IGF-I-stimulated IRS-1 phosphorylation is modulated by {alpha}vbeta3-integrin. A: representative immunoblots of the time-dependent IGF-I-stimulated IRS-1(Tyr632) phosphorylation in control and echistatin-treated human intestinal muscle cells. B: IGF-I stimulates time-dependent IRS-1(Tyr632) phosphorylation that is inhibited in the presence of the {alpha}vbeta3-integrin antagonist echistatin. Quiescent muscle cells were treated for 0–30 min with 100 nM IGF-I in the presence and absence of 100 nM echistatin. IRS-1(Tyr632) phosphorylation was measured by immunoblotting for IRS-1(Tyr632) using a phosphospecific antibody. Results were expressed in relative densitometric units and normalized to total (phosphorylated plus unphosphorylated) IRS-1 levels. Values represent the means ± SE of 4 separate experiments. *P < 0.05 vs. control.

 
Role of {alpha}vbeta3-integrin in IGF-I-stimulated Erk1/2 phosphorylation.

As noted above, we have previously shown that endogenous IGF-I stimulates muscle cell growth by activation of distinct signaling pathways downstream of the IGF-IR that lead to Erk1/2 and p70S6 kinase activation (20, 22). The role of {alpha}vbeta3-integrin in modulating IGF-I-stimulated Erk1/2 activation was examined in the presence of the {alpha}vbeta3-disintegrin echistatin and in the presence of the {alpha}vbeta3-integrin ligand fibronectin.

Incubation of quiescent muscle cells with 100 nM IGF-I for time periods of 0–30 min caused phosphorylation of Erk1/2(Thr202/Tyr204) that was maximal within 10 min and declined after 30 min (Fig. 7). In the presence of fibronectin (5 µg/cm2 cultureware surface area), IGF-I-stimulated maximal Erk1/2(Thr204/Tyr204) phosphorylation was significantly increased and phosphorylation was sustained at maximal levels for 30 min (Fig. 7). In contrast, in the presence of echistatin, IGF-I-stimulated Erk1/2(Thr202/Tyr204) phosphorylation was significantly inhibited at all time points measured (Fig. 7). These results implied that the endogenous {alpha}vbeta3-ligands produced by intestinal muscle cells vitronectin and fibronectin (Fig. 1) modulate the effects of IGF-I on Erk1/2 activation.


Figure 7
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  		Fig. 7. IGF-I-stimulated Erk1/2 phosphorylation is modulated by the {alpha}vbeta3-integrin. A: representative immunoblots of time-dependent IGF-I-stimulated Erk1/2 phosphorylation in control and fibronectin- and echistatin-treated human intestinal smooth muscle cells. B: IGF-I elicits time-dependent Erk1/2 phosphorylation. In the presence of the {alpha}vbeta3-integrin ligand fibronectin, IGF-I-stimulated Erk1/2 phosphorylation is enhanced at all time periods. In the presence of the {alpha}vbeta3-integrin antagonist echistatin, IGF-I-stimulated Erk1/2 phosphorylation is inhibited at all time periods. Quiescent muscle cells were treated for 0–30 min with 100 nM IGF-I. Fibronectin (5 µg/cm2) was used as a ligand of the {alpha}vbeta3-integrin, and echistatin (100 µM) was used as a selective {alpha}vbeta3-integrin antagonist. Erk1/2 phosphorylation was determined by immunoblot analysis using an Erk1/2(Thr202/Tyr204) phosphospecific antibody. Results were expressed in relative densitometric values normalized to total (phosphorylated plus unphosphorylated) Erk1/2 levels. Values represent the means ± SE of 3–4 separate experiments. *P < 0.05 vs. control.

 
Role of {alpha}vbeta3-integrin in IGF-I stimulated p70S6 kinase phosphorylation.

The role of {alpha}vbeta3-integrin on IGF-I-stimulated p70S6 kinase phosphorylation was also examined. p70S6 kinase(Ser389) phosphorylation was used as a measure of activation; multiple residues are phosphorylated during p70S6 kinase activation, but Ser389 phosphorylation is most closely correlated with its kinase activity (22, 22). As shown previously, incubation of quiescent muscle cells with 100 nM IGF-I increased the phosphorylation of p70S6 kinase(Ser389) in a time-dependent fashion that was maximal after 30 min (Fig. 8). In the presence of fibronectin (5 µg/cm2 cultureware surface area) IGF-I-stimulated p70S6 kinase(Ser389) phosphorylation was significantly increased (Fig. 8). In contrast, in the presence of the {alpha}vbeta3-specific disintegrin echistatin, IGF-I-stimulated p70S6 kinase(Ser389) phosphorylation was significantly inhibited at all time points (Fig. 8).


Figure 8
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  		Fig. 8. IGF-I-stimulated p70S6 kinase phosphorylation is modulated by the {alpha}vbeta3-integrin. A: representative immunoblots of time-dependent IGF-I-stimulated p70S6 kinase phosphorylation in control and fibronectin- and echistatin-treated human intestinal smooth muscle cells. B: IGF-I elicits time-dependent p70S6 kinase phosphorylation. In the presence of the {alpha}vbeta3-integrin ligand fibronectin, IGF-I-stimulated p70S6 kinase phosphorylation is enhanced at all time periods. In the presence of the {alpha}vbeta3-integrin antagonist echistatin, IGF-I-stimulated p70S6 kinase phosphorylation is inhibited at all time periods. Quiescent muscle cells were treated for 0–30 min with 100 nM IGF-I. Fibronectin (5 µg/cm2) was used as a ligand of the {alpha}vbeta3-integrin, and echistatin (100 µM) was used as a selective {alpha}vbeta3-integrin antagonist. p70S6 Kinase phosphorylation was determined by immunoblot analysis using a p70S6 kinase(Ser389) phosphospecific antibody. Results were expressed in relative densitometric values normalized to total (phosphorylated plus unphosphorylated) p70S6 kinase levels. Values represent the means ± SE of 3–4 separate experiments. *P < 0.05 vs. control basal values.

 
Role of {alpha}vbeta3 in IGF-I-stimulated proliferation.

We have previously shown that endogenous IGF-I stimulates human intestinal smooth muscle cell proliferation by activation of the IGF-IR tyrosine kinase (21). The participation of endogenous {alpha}vbeta3-integrin ligands in modulating IGF-I-stimulated activation of the IGF-IR tyrosine kinase IRS-1 and the signaling intermediates Erk1/2 and p70S6 kinase suggested that endogenous integrin ligands might modulate IGF-I-stimulated proliferation. This possibility was examined by measuring the proliferative response to IGF-I (100 nM) in the presence of the {alpha}vbeta3-integrin antagonist echistatin (100 nM) or the {alpha}vbeta3-integrin ligand fibronectin (5 µg/cm2 cultureware surface area).

Basal levels of [3H]thymidine incorporation were augmented 50 ± 14% by fibronectin and inhibited 54 ± 11% by echistatin (Fig. 9). In the presence of fibronectin, IGF-I-stimulated proliferation was increased by 400 ± 30% above that elicited by 100 nM IGF-I under control conditions (440 ± 29% above basal levels). In contrast, in the presence of echistatin, IGF-I-stimulated proliferation was inhibited by 47 ± 10% from that observed under control conditions. These results, taken together with the finding that human intestinal smooth muscle cells produce vitronectin and fibronectin, suggest that occupancy of {alpha}vbeta3-integrin by endogenous {alpha}vbeta3-integrin ligands modulate IGF-I-stimulated growth of human intestinal smooth muscle cells.


Figure 9
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  		Fig. 9. IGF-I-stimulated growth is modulated by {alpha}vbeta3-integrin. IGF-I stimulates [3H]thymidine incorporation in human intestinal smooth muscle cells. In the presence of the {alpha}vbeta3-integrin ligand fibronectin, IGF-I-stimulated proliferation is augmented. In the presence of the {alpha}vbeta3-integrin echistatin, IGF-I-stimulated proliferation is inhibited. Quiescent muscle cells were treated for 24 h with 100 nM IGF-I. Fibronectin (5 µg/cm2) was used as a ligand of the {alpha}vbeta3-integrin, and echistatin (100 µM) was used as a selective {alpha}vbeta3-integrin antagonist. Results were expressed as %basal [3H]thymidine incorporation in control cells. Values represent the means ± SE of 4 separate experiments. *P < 0.05 vs. control basal values. **P < 0.05 vs. control IGF-I values.

 

   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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Endogenous IGF-I regulates the growth of human intestinal smooth muscle cells by jointly stimulating proliferation and inhibiting apoptosis (18, 19). These effects are mediated by IGF-I-stimulated activation of its cognate receptor tyrosine kinase. Binding of IGF-I to the IGF-IR tyrosine kinase results in rapid, time-dependent phosphorylation of specific residues within the ATP-binding domain, the autophosphorylation domain, and in distinct domains that couple to the primary substrates of the activated IGF-IR tyrosine kinase (11). Although phosphorylation of the IGF-IR by IGF-I is rapid (maximal within 2 min), it is also transient and declines to suprabasal levels within 30 min. The biphasic kinetics of receptor activation and rapid deactivation suggest that dephosphorylation of the receptor might also be a regulated process. The present paper shows that occupancy of the {alpha}vbeta3-integrin by its endogenous ligands regulates the extent and duration of IGF-I-stimulated IGF-IR activation by determining the rate of recruitment of SHP-2 phosphatase to the activated IGF-IR.

We have previously shown that IGF-I is an autocrine growth factor of human intestinal smooth muscle cells (19). The present paper shows that the {alpha}V- and beta3-integrin subunits and the {alpha}vbeta3-ligands vitronectin and fibronectin are expressed by these cells. The evidence supporting a role for {alpha}vbeta3-integrin-dependent modulation of IGF-I-stimulated IGF-IR activation and proliferation in human intestinal smooth muscle cells can be summarized as follows: 1) IGF-I-stimulated IGF-IR phosphorylation is augmented by the {alpha}vbeta3-integrin ligand fibronectin and inhibited by the {alpha}vbeta3-selective disintegrin echistatin; 2) SHP-2 phosphatase recruitment to the IGF-IR is accelerated in the presence of echistatin, and maximal association is correlated in time with IGF-IR dephosphorylation; 3) IGF-I-induced recruitment of IRS-1 to the activated IGF-IR and IRS-1 phosphorylation is inhibited by echistatin; 4) IGF-I-induced activation of Erk1/2 and p70S6 kinase is augmented by fibronectin and inhibited by echistatin; and 5) basal and IGF-I-stimulated proliferation of intestinal smooth muscle cells are inhibited by echistatin and augmented by fibronectin.

In human intestinal smooth muscle cells and porcine vascular smooth muscle cells, occupancy of the {alpha}vbeta3-integrin is required for optimal activation of IGF-IR tyrosine kinase by IGF-I. This paper and work by Clemmons and colleagues (7, 26, 27, 29) show that in the presence of peptide and nonpeptide antagonists of the {alpha}vbeta3-integrin, the duration and extent of IGF-I-stimulated IGF-IR phosphorylation are significantly reduced and result in decreased IGF-I-stimulated IGF-IR tyrosine kinase activation, proliferation of intestinal and vascular muscle cells, and migration and protein synthesis in vascular muscle cells. {alpha}vbeta3-Integrin-dependent modulation of these events is mediated by its ability to regulate the rate of recruitment of the protein tyrosine phosphatase SHP-2 to the activated IGF-IR (27). When activation of {alpha}vbeta3-integrin is blocked, SHP-2 is recruited more rapidly to specific phosphotyrosine motifs of the activated IGF-IR tyrosine kinase. SHP-2 specifically binds to phosphorylated tyrosine residues that are followed by a specific motif YXXL/I and causes dephosphorylation of these phosphotyrosine residues (6, 23). After dephosphorylation, SHP-2 looses affinity and disassociates.

In addition to its role in regulating the extent and duration of IGF-I-stimulated IGF-IR tyrosine kinase activation, protein tyrosine phosphatase SHP-2 can be recruited to other activated growth factor receptors (PDGF, insulin, and epidermal growth factor) that possess these specific phosphotyrosine-containing motifs, where it functions similarly to regulate the activity of these receptor tyrosine kinases (3, 13). SHP-2 binds to Tyr771 of the PDGF-beta receptor, elicits receptor tyrosine kinase dephosphorylation, and diminishes signaling to the Ras/Erk pathway (10). SHP-2 also binds to Tyr992 of the EGF receptor, negatively regulating ligand-dependent signaling (1). In addition to its affinity for phosphotyrosine residues in growth factor receptors, SHP-2 can also modulate signaling via IL receptors through their tyrosine residues. SHP-2 binds to Tyr759 of the IL-6 signal transducing receptor subunit gp130 and attenuates IL-6-stimulated Jak/STAT activation (24) The canonical immunoreceptor tyrosine-based inhibitory motif (I/VxYxxL), which is also found in the IL-4 receptor, is a target of protein tyrosine phosphatase SHP-2 and modulates IL-4-stimulated proliferation (16).

The fundamental role of IGF-I in regulating smooth muscle growth and development throughout the body, and in the intestine in particular, is highlighted by investigations using mutant mice under- or overexpressing IGF-I. Mice overexpressing IGF-I exhibit hyperplasia of the intestinal smooth muscle layers (30). Mice with a Cre-LoxP targeted disruption of hepatic IGF-I production have greatly diminished levels of circulating IGF-I, yet their intestinal muscle layers develop normally (35). These results imply that the mechanisms regulating IGF-I-stimulated activation of its cognate receptor in intestinal smooth muscle are autocrine and/or paracrine in nature and are important determinates of smooth muscle growth.

The potential clinical significance of the mechanisms investigated in this paper occurs in the setting of chronic intestinal inflammation. Associated with chronic intestinal inflammation of Crohn's Disease are increased levels of the {alpha}vbeta3-integrin ligands vitronectin and fibronectin (2). Endogenous expression of IGF-I by inflamed intestinal muscle is also significantly increased (39). The concomitant increased activation of {alpha}vbeta3-integrin and IGF-IR tyrosine kinase of human intestinal smooth muscle can result in increased activation of IGF-I-stimulated signaling pathways leading to increased muscle cell proliferation via Erk1/2 and p70S6 kinase (22), and inhibition of apoptosis via GSK-3beta (18). The result of these events that jointly stimulate intestinal smooth muscle cell proliferation and inhibition of apoptosis could lead to the muscle hyperplasia and stricture formation in patients with Crohn's Disease.


   GRANTS
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 ABSTRACT
 METHODS
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 GRANTS
REFERENCES
 
This work was supported by Grant DK-49691 from the National Institute of Diabetes and Digestive and Kidney Diseases.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. F. Kuemmerle, Division of Gastroenterology, Medical College of Virginia Campus, Virginia Commonwealth Univ., P.O. Box 980341, Richmond, VA 23298–0341 (e-mail: jkuemmerle{at}hsc.vcu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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